Enzymatic Catalysis In Non-aqueous Media Of Phospholipase A1and Its Immobilization

Soybean phosphatidylcholine (PC) is widely used in food, medicine, cosmetics,agricultural products and other industries, and the Enzymatic Modification of PChas been studyed more in recent years. In this paper, PC was hydrolyzed completelyby phospholipase in non-aqueous media and converted into the final productglycerolphospho choline (GPC). The construction of the reaction system, thepreliminary separation and purification of the product, the establishment of theanalytical method, process optimization, the recycling of the organic solvent, thereaction mechanism and enzyme immobilization were exploratory studyed.Firstly,the W/O microemulsion system was built with the substrate PC assurfactant, butanol as the cosurfactant, isooctane as the oil phase and the acetatebuffer as the aqueous phase. The composition of the microemulsion waspreliminary determined as follows, PC: isooctane: butanol: water=1:6.3:0.9:0.5,including PC unit of mass (g) and the other unit of volume (mL). In this reactionsystem, Phospholipase was used as a catalyst and the acid-base titration was used todetermine the end point of the reaction. After the reaction, the reaction system wastreated by the preliminary separation and purification-extraction, then the GPCsample could be qualitatively and quantitative determined by TLC and HPLCrespectively. The yield of GPC sample is85%by HPLC at this situation.Secondly,the process of enzymatic preparation of GPC wasoptimized. Optimum temperature40℃; the optimum pH5.0; Ca2+having no effecton the yield of the product; enzyme and buffer volume ratio of1:2before substratewas added; the initial substrate concentration was0.278g/mL, that means the initialreaction system was turned to as follows, PC: isooctane: n-butanol: aqueous phase=2:6.3:0.9:0.5; additional substrate was added with the concentration of0.437g/mL，50%of initial substrate amount; a certain amount of buffer needed toadd when adding the substrate and the adding amount was based on PC: isooctane:n-butanol: aqueous phase=1:2:0.286:0.25; the adding time was1.5h after theinitial reaction time and the total reaction time was6h. After process optimization,GPC yield increased to96.5%. The reaction reagents was firstly separated byvacuum rotary evaporator,then GPC sample was extracted from the rest fluid, inthis way, the reagents and the extractant could be recycled respectively. The extractant dichloromethane and methanol/water were recycled by vacuum rotaryevaporator; the volume ratio of the reagents isooctane and n-butanol was measuredusing gas chromatography external standard quantitative method, and then preparedto the original ratio of7:1, re-used in the next batch reaction. During the catalyticprocess, the fact that the intermediate product LPC was generated during reactionand finaly converted into GPC by acylmigration was confirmed by TLC method.Finally,the immobilization of phospholipase was studyed. By comparative studyon the three immobilization methods: glutaraldehyde cross-linking,adsorption-embedding and adsorption, it was found the enzyme immobilized byadsorption had the best catalytic effect. Then, the four carriers diatomite, cationexchange resin001*7(732), Seplite resin LX-1000EP, Seplite resinLX-1000NHF,were studyed to compare the adsorption immobilizationeffect. LX-1000NHF was the optimal carrier，and its catalytic reaction had theshortest catalytic time and highest yield of product. Immobilized conditions: carrierand enzyme ratio of5:2(m/v), the adsorption time of5h, the adsorptiontemperature of room temperature. Km values of immobilized enzyme and freeenzyme were0.705g/ml and0.184g/ml respectively. Catalytic conditions: substrateand immobilized enzyme mass ratio of5:3, the optimum temperature of60℃,substrate and water content ratio of5:2(m/v). The GPC yields of the first twobatches were98.6%and87.6%respectively, which were higher than the yieldcatalyzed by free enzyme (85%).A feasible method was provided by the process for the preparation ofglycerophosphatide choline. It provides the foundation for its industrial applicationand a reliable theoretical basis for the enzymatic modification of lecithin.